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Protoporphyrin PPIX-high-yielding shewanella genetic engineering bacterium and construction method thereof

A technology of Shewanella and construction method, applied in the field of genetic engineering, can solve the problems of high price, long production cycle and high extraction cost, and achieve the effect of high price and high quality

Inactive Publication Date: 2019-02-01
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of chemical synthesis has cumbersome steps, low yield, high cost and high price.
Heme is mainly obtained from animal blood. The same method is cumbersome and requires a lot of chemical reagents.
With the advancement and development of technology, although the extraction method has been improved, it still relies on the catalysis and chemical reaction of chemical reagents, but the following issues need to be considered in order to be truly applied in production: 1. Reduce costs
The existing method has high cost and single product, and fails to co-produce other products; 2. In the process of extraction, the use of organic solvents that are highly toxic and difficult to recycle should be avoided as much as possible to achieve environmentally friendly production
The common shortcomings of previous methods are low product purity, high extraction cost, long production cycle, and difficult solvent recovery, etc.
And because the hemoglobin in the hemoglobin of red blood cells is connected with globin, it is difficult to extract high-purity products from blood

Method used

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  • Protoporphyrin PPIX-high-yielding shewanella genetic engineering bacterium and construction method thereof
  • Protoporphyrin PPIX-high-yielding shewanella genetic engineering bacterium and construction method thereof
  • Protoporphyrin PPIX-high-yielding shewanella genetic engineering bacterium and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of Gene Knockout Suicide Plasmid

[0031] The upstream and downstream parts of the hemH1 and hemH2 genes were connected and introduced into the multiple cloning site of the recombination-integrated suicide plasmid PDS3.0 to obtain the suicide knockout plasmid p△hemH1 containing the hemH1 gene and the suicide knockout plasmid containing the hemH2 gene, respectively. Plasmid pΔhemH2 was removed. The specific operation is as follows:

[0032] According to the whole genome sequence (GCA_000146165.2) of wild-type Shewanella (S.oneidensis) MR-1 in the GenBank database, primers for knocking out hemH1 were designed respectively. The primer sequences are as follows:

[0033] hemH1-sacI-3o:GTgagctcGATGGCGGCAAACGGTATTA (with SacI restriction site)

[0034] hemH1-sacI-3i:agagacgacctaagccagtcGACGACCCGAGGATCACTTA

[0035] hemH1-sacI-5o:gactggcttaggtcgtctctCCAAGGCGTGAAAAGTGTCG

[0036] hemH1-sacI-5i:CAgagctcTCGTCGGCCAAAATAGAATA (with SacI restriction site) ...

Embodiment 2

[0048] Example 2: Construction of double gene knockout engineering strain MR-1ΔhemH1hemH2

[0049] Shewanella onedensis MR-1 was originally isolated from the anaerobic sediments of Oneida Lake in New York State, USA (Bacterial manganese reduction and growth with manganese oxide as the sole electron-acceptor. Science. 1988; 240(4857):1319–21.), is a Gram-negative bacterium belonging to a subgroup of the γ-proteobacteria, with facultative anaerobic characteristics, and the optimum growth temperature is around 30°C. As a model strain of Shewanella, it can be obtained in many laboratories, and can also be purchased from the BioVector plasmid vector strain cell gene collection center.

[0050] The principle and flow diagram of hemH1 and hemH2 gene knockout are as follows figure 2 As shown, the specific operation method is as follows:

[0051] First, the constructed gene knockout suicide plasmid p△hemH1 was transferred into the auxotrophic strain E.coliWM3064. The suicide plasmid...

Embodiment 3

[0055] Example 3: Chemical analysis of the red substance produced by engineering strain MR-1ΔhemH1hemH2

[0056]We qualitatively and quantitatively analyzed the red substance produced by the double gene knockout strain MR-1ΔhemH1hemH2 by ultraviolet spectrophotometry and mass spectrometry respectively. ,13,17-tetramethyl-21II,23II-porphine 2,18dipropanoieacid) standard sample and the red substance produced by double gene knockout engineering strain MR-1ΔhemH1hemH2 were dissolved in 90% acetone solution and 10% 0.1mol / L NH 4 In OH, use a UV spectrophotometer (BiowaveII, WPA), measure the absorption peaks of the standard sample and our sample to be tested with a quartz cuvette in the wavelength range of every 10nm, so as to measure the absorption peak curve, and read its The maximum absorption peak, and found that the maximum absorption peak appears at the same time around 405nm (such as Figure 5 ). And the result of mass spectrometry analysis also shows that the mass spectro...

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Abstract

The invention discloses a protoporphyrin PPIX-high-yielding shewanella genetic engineering bacterium and a construction method thereof. Suicide-type knockout plasmids p[delta]hemH1 and p[delta]hemH2 are constructed respectively. The suicide plasmids enter host shewanella by a conjugation way, and ferrous chelatase genes hemH1 and hemH2 of shewanella are subjected to double knockout by a homologousrecombination way; meanwhile, chlorhematin is added by an exogenous way to maintain the growth of the engineering bacterium, a large amount of protoporphyrin PPIX is accumulated and released to the outside of cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Shewanella engineering bacterium for double knockout of ferrochelatase genes hemH1 and hemH2, and the engineering bacterium can be used to produce protoporphyrin (PPIX). Background technique [0002] Protoporphyrin IX (PPIX) is a hydrophobic organic compound consisting of 4 pyrrolyl groups and a porphyrin ring with a conjugated system. Its unique ring structure in the cell makes it easy to coordinate with some metal ions to form metalloporphyrin compounds, such as iron porphyrin, cobalt porphyrin, copper porphyrin, manganese porphyrin, so it has various biological functions . Among them, protoporphyrin IX chelates iron ions to form heme (iron protoporphyrin IX, heme), and then combines with some proteins to form hemoglobin, peroxidase, cytochrome, etc. important role in drug metabolism. Therefore, protoporphyrin IX, as the precursor of hemoglobin synt...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P17/18
CPCC12N9/88C12N15/74C12P17/182C12Y499/01001
Inventor 邱东茹戴景程周集中
Owner INST OF AQUATIC LIFE ACAD SINICA
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